发信人: shawl (a real tough cookie), 信区: Biology
标 题: Re: 请教免疫荧光
发信站: Unknown Space - 未名空间 (Thu Apr 1 12:17:02 2004) WWW-POST

This is what i would do if i were you:

1. Check if the 2nd ab cross-react with the species of your tissue sample
2. Use cold acetone to permealize and fix the tissue in step2
3. decrease the 1st ab concentration,and try O/N incubation in 4 degree
4, mix 2 2nd ab and spin at max speed for 2min. Sometimes, Texas red and FITC
form small particle precipitates.

I think the wash is enough in your protocol.

Good luck



【 在 moses (Moses) 的大作中提到: 】
: 用如下程序做组织冰冻切片染色，结果背景高，特异染色信号强度低，还怎么改进呢？
谢
: 谢！
:
: 1. Wash in PBS for 5 min;
:
: 2. Wash in PBS+0.1%Sapotonin for 10 min;
:
: 3. Wash in PBS for 2 X 5 min;
:
: 4. Block with 5% goat serum + 5% mouse serum for 1hr at RT;
:
: 5. Dilute primary Ab with blocking buffer and incubate for 1hr at RT;
:
: 6. Wash slides in PBS +0.1%Tween-20 for 3 X 10 min;
:
: 7. Dilute secondary Ab with blocking buffer and incubate for 1hr at RT;
:
: 8. Wash slides in PBS +0.1%Tween-20 for 3 X 10 min;
:
: 9. Mount and coverslip.
:
:


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